Vaccine formulation potentiated by the combination of a dna and an antigen

ABSTRACT

Formulation of vaccine antigens, containing as main components: a-) one or several DNA expressing one or several proteins in the immunized individuals and b-) a viral antigen, in appropriate proportions. The novelty of the invention is given by the enhancing effect of at least one component on the immune response generated against the other one.  
     Development of new formulations, minimizing the number of components that enhance and diversify the spectrum of immune response against different pathogenic entities and generating combined vaccines against pathogens. These formulations can be applied in the pharmaceutical industry for preventive and-or therapeutic use in human.

FIELD OF THE INVENTION

[0001] The present invention is related with the branch of the medicine,particularly with a new formulation of vaccine antigens. The technicalobjective of the present invention is the development of new vaccineformulations, minimizing the number of components that are able toinduce an enhanced and diverse immune response through the interactionamong them. Additionally, the development of combined vaccineformulations is approached in order to increase the immune responseinduced against the co-administered antigens.

PREVIOUS TECHNIQUE

[0002] Several obstacles exist for the obtaining of an effective vaccineagainst the HCV. Because its RNA nature, HCV can quickly mutate inadaptation to the environment. This contributes to the high diversity ofsequences of the multiple viral isolates identified in the world. Thebiggest heterogeneity concentrates on the hypervariable region I of theHCV E2 protein, where a possible neutralizing epitope has beendescribed. The HCV causes persistent infection in spite of the existenceof an active immune response (Lechmann et al., Semin Liver Dis 2000, 20,211-226). Neither an animal model, nor an in vitro culture system forthe efficient replication of the virus and the study about theoccurrence of neutralizing antibodies exist. The immunologic patternsassociated with the progression of the illness or with the protectionhave not been defined. It is probable that potent, multispecific andlong-lasting both, humoral and cellular immune responses are requiredfor the resolution of the infection (Lechmann et al., Semin Liver Dis2000, 20, 211-226). Several approaches have been used to develop avaccine against the HCV. Recombinant proteins, synthetic peptides, viruslike particles, DNA vaccines and live-viral vectors are the most widelyevaluated.

[0003] The development of a vaccine based on protein subunits was one ofthe first strategies evaluated for the HCV because for severalflaviviruses, antibodies directed against surface proteins can conferprotection. Some variants based on the HCV structural antigens haveachieved limited protection against the virus in animal models. Such itis the case of the chimpanzees immunized with EI-E2 heterodimers. Sevenchimpanzees were vaccinated, five were protected and two developed aself-limiting disease (Choo et al., PROC NATL ACAD SCI USES 1994, 91,1294-1298). This protection has been correlated with the presence ofantibodies (Abs) able to inhibit the E2 binding to human cells (Rosa etal., PROC NATL ACAD SCI USES 1996, 93, 1759-1763).

[0004] The recombinant E1 protein from an isolate of the genotype 1 bwas purified as homodimers self-associating in particles of 9 nmdiameter, approximately (Maertens et al., Records Gastroenterol Belg2000, 63, 203). Two chimpanzees chronically infected with HCV received 9doses of 50 μg of the recombinant E1 protein. The vaccination improvedthe hepatic histology and determined the disappearance of the viralantigens of the liver. Vaccination with recombinant E1 protein alsoreduced the levels of alanine aminotransferase (ALT). Although thelevels of viral RNA in serum didn't change during the treatment, theliver inflammation and the levels of viral antigens increased aftertreatment. An association was observed between the high levels ofantibodies against E1 and the improvement of the illness (Maertens etal., Records Gastroenterol Belg 2000, 63, 203).

[0005] Particularly, the formation of virus-like particles fromrecombinant proteins and their employment as vaccines is very attractivebecause these structures frequently simulate viral properties. This kindof particles, obtained from insect cells infected with a recombinantbaculovirus containing the sequence of the HCV structural antigens, havebeen able to generate both humoral and cellular immune response againstthese antigens (Baumert et al., Gastroenterology 1999, 117, 1397-407;Lechmann et al., Hepatology 1999, 30, 423-429). Although the resultsobtained with vaccines based on protein subunits are encouraging, theimmune response induced by these variants is mainly humoral, short-termand isolate-specific.

[0006] On the other hand, different recombinant viral vectors have beenevaluated in the development of a recombinant vaccine against the HCV.Particularly, recombinant adenoviral vectors are interesting candidatesdue to their liver tropism, their power to induce both humoral andcellular immunity, and the feasibility for oral or systemic delivery.Adenoviruses containing the DNA encoding sequence for the HCV structuralproteins induce an antibody response against each one of these proteins(Makimura et al., Vaccine 1996, 14, 28-36). Moreover, after immunizationin mice with recombinant adenoviruses for C and E1, a specific CTLresponse is detected against these antigens (Bruna-Romero et al.,Hepatology 1997, 25, 470-477). Although these results have beenencouraging, the recent problems with the use of recombinantadenoviruses in gene therapy have raised doubts about their employmentin humans. Other recombinant viruses, like vaccinia, canary-pox andfowl-pox, containing different HCV genes have induced strong CTL andT-helper immune responses in mice (Shirai et al., J Virol 1994, 68, 3334-3342; Large et al., J Immunol 1999, 162, 931-938). However, theserecombinant viruses, as well as other variants of alpha virus like theSemliki Forest Virus are also affected by regulatory issues and securityconcerns related with their application.

[0007] The identification of several epitopes for CD4+ and CD8+ T cellsin the HCV polyprotein, which could be important in the viralelimination, support the evaluation of synthetic peptides as vaccinecandidates against this pathogen. Different peptides, lipidated or not,containing epitopes of C, NS4 and NS5, have induced a strong CTLresponse in mice (Shirai et al., J Infect Dis 1996, 173, 24-31; Hiranumaet al., J Gene Virol 1999, 80, 187-193; Oseroff et al., Vaccine 1998,16,823-833).

[0008] Another strategy used to develop a vaccine against the HCV isbased in the possibility of generating Abs against linear epitopes. Thisalternative has been evaluated basically to generate Abs against theHVR-l of the HCV, with some encouraging results in rabbits andchimpanzees (Esumi et al., Arch Virol 1999, 144, 973-980; Shang et al.,Virology 1999, 258, 396-405). Quasi-species is the main problem ofselecting the HVR-l as the target for a vaccine against the HCV.

[0009] The main obstacle for the peptide vaccines is that those peptideswithout epitopes for helper T cells can be poorly immunogenics.Moreover, the effectiveness of a vaccine is frequently based on theinduction of specific immune response against a wide range of differentantigens. These limitations are important weaknesses of this strategy.

[0010] The DNA immunization is one of the most recent strategies invaccine development. A DNA vaccine consists on a purified plasmidcontaining the sequence coding for an antigen of interest, under thecontrol of a functional transcriptional unit in eucariotic cells. Afterinjection of the plasmid in muscle or the skin, the plasmid is taken upby host cells and the antigen is expressed intracellularly. Theexpression of the encoded antigens in the host cells is one of the majoradvantages of this methodology because is similar to viral naturalinfections. The simplicity to manipulate the DNA, together with the DNAstability that makes possible a relatively cheap large-scale productionof DNA, is other advantage of DNA vaccination.

[0011] The immune response induced with this kind of vaccines can bemodulated by co-immunization with molecules or genes coding forco-stimulatory molecules like cytokines. The genetic constructs can bemodified, by insertions or deletions of transmembrane domains, signalsequences for secretion, or other types of residues affecting theintracellular trafficking and processing of the antigen.

[0012] Particularly, the DNA immunization has been largely studied inthe development of vaccines against the HCV. Different expressionvectors encoding full-length or truncated variants of the HCV capsidprotein have been generated (Lagging et al., J Virol 1995, 69,5859-5863; Chen et al., Vaccine Res 1995, 4, 135-144). Other constructsalso include the HCV 5′ non-translated region (Tokushige et al.,Hepatology 1996, 24, 14-20). Plasmids expressing fusion variants to thehepatitis B virus (HBV) surface antigen or other envelope antigens ofthe HBV have been evaluated (Major et al., J VIROL 1995, 69, 5798-5805).Immunization with these plasmids has generally induced positive CTL andlymphocyte proliferative response.

[0013] The HCV envelope proteins have also constituted targets ofinterest for this type of technology. In the case of the HCV E2, thehumoral response seems to be mainly directed to the HVR-1 (Lee et al.,Mol Cells 1998, 8, 444-451). Immunization with plasmids expressingintracellular or secreted variants of the E1 and E2 proteins hasrendered similar immune response (Lee et al., J VIROL 1998, 72,8430-8436). The inoculation with bicistronic plasmids expressing theGM-CSF and the HCV E1 or E2 proteins increased both humoral and cellularimmune response. Recently, the use of bicistronic plasmids expressingthe E1 and E2 proteins were generated to investigate the influence ofheterodimer formation between these proteins in vivo on the immuneresponse induced after DNA immunization. When heterodimers were formed,the antibody response against HCV E1 and E2 proteins was not obtained.In sharp contrast, high-level antibody titers, directed to both linearand conformational epitopes, were obtained after immunization withplasmids expressing truncated variants of E1 and E2. Therefore, it seemsnecessary to avoid the heterodimers formation to obtain a strongantibody response when constructs including these antigens are evaluated(Fournillier et al., J VIROL 1999, 73, 497-504).

[0014] The non structural proteins have also been evaluated by thistechnology. Good results were obtained when the region coding for theC-terminal domain of the NS3 protein was included in a vector thatallows the simultaneous and independent expression of this domain andthe IL-2 (Papa et al., Res Virol 1998, 149, 315-319). The NS4 and NS5proteins also generate Abs and CTL responses by this immunizationstrategy (Encke et al., J IMMUNOL 1998, 161, 4917-4923). Recently, theuse of a construction coding for GM-CSF and the non structural proteinsof the virión (NS3, NS4 and NS5) induced a potent Ab response and apotentiated lymphoproliferative response against each non structuralprotein (Cho et al., Vaccine 1999,17, 1136-1144).

[0015] In general, the effective expression of different HCV antigens,as well as the generation of anti-HCV Abs in levels ranging from 1:100to 1:100 000 according to the combination in study, has been reportedfor different DNA constructs (Inchauspe et al., Vaccine 1997, 15,853-856). Additionally, the development of specific CTL and lymphocyteproliferative response has been demonstrated (Inchauspe et al., DNA ANDCELL BIOLOGY 1997, 16, 185-195). However, efforts are required toimprove this methodology in order to generate stronger both humoral andcellular response against different proteins of the HCV. Thus, somevariants like liposomes (Gramzinski et al., Mol Medicine 1998, 4,109-118) and saponin QS-21 (Sasaki et to the., J Virol 1998, 72,4931-4939) have been evaluated to increase the immune response inducedafter DNA vaccination. The dendritic cells as biological adjuvants havebeen also studied in DNA immunization. Dendritic cells (CD) derived offormer genetically modified mouse bone marrow to express tumor antigens,by using viral vectors (Specht et al., J Exp Med 1997, 186, 1213-1221;Brossart et al., J Immunol 1997, 158, 3270-3276; Song et al., J Exp Med1997, 186, 1247-1256), or RNA (Boczkowski et to the., J Exp Med 1996,184, 465-472), have demonstrated their capacity to promote T cellresponse specific for tumor antigens, and prophylactic immunity mediatedby cells against tumors in mouse.

[0016] At the present, the improvement of vectors for DNA immunization,including the insertion of CpG motifs to increase the immune responseagainst the expressed antigens (Hasan et al., J Immunol Meth 1999,229,1-22), and the DNA delivery systems is crucial to overcome thelimitations of this technology. Due to the challenges that outlines theHCV infection, and to the absence of a clear definition about theimmunologic parameters correlating with the protection against thispathogen, it is possible that an effective vaccine against the HCV shallrequire a multispecific approach stimulating several aspects of theimmune response. The solution of this problem is probably in thecombination of several vaccination strategies explored until the moment.Particularly, immunization schedules that combine a prime dose with aDNA vaccine and a booster dose with recombinant proteins or viralvectors (Hu et al., Vaccine 1999, 17, 3160-3170; Pancholi et al., JInfect Dis 2000, 182, 18-27) have been evaluated with results that,although positives, require additional investigations to demonstrate ifthe prime-boost strategies can really induce a protective immunityagainst the HCV.

[0017] Additionally, for the hepatitis B model, a vaccine compositioncomprising the complex formed by the hepatitis B surface antigen, anantibody specific for this antigen, and a DNA vaccine expressing forthis antigen has been evaluated (Wen et al., U.S. Pat. No. 6,221,664,1998). This formulation allowed the antigen presentation by differentmeans and a quick induction of immune response that resulted superiorregarding to the one generated by the individual variants.

[0018] In the present invention, a vaccine formulation comprising ascomponents only a protein antigen and a plasmid expressing one orseveral proteins, acting at least one of them as adjuvant of the otherone, is described. Particularly, the capsid antigen of the hepatitis Cor B virus, and a plasmid expressing individual or polyprotein variantsof the HCV E1 protein, are evaluated. Contrary to the compositionpreviously described for the hepatitis B model, the presence ofantibodies in the formulation is not required to generate theenhancement of the immune response, thus reducing the number ofcomponents required. Additionally, the biggest flexibility in thevaccine composition also allows generating simultaneously potent immuneresponses against different antigens.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides the composition and methods toimmunize an individual in a prophylactic or therapeutic way against theHCV and the HBV, as well as their combination. It is reported for thefirst time a vaccine formulation having as components: (a) a DNA thatexpresses a protein variant that includes regions of the E1 antigen ofthe HCV envelope and (b) a protein antigen of the HCV or HBV, inappropriate proportions. The novelty of the invention is given by theadjuvant effect of at least one component on the immune responsegenerated against the other one. Antigens coded by the geneticconstructs and expressed by the host cells, as well as the proteinantigen comprising the vaccine the formulation, are interesting targetsto generate an immune response against the HCV and the HBV. Thus, theimmune response can be directed against a wide spectrum of importantantigens.

[0020] The vaccine formulation includes a DNA enhancing the immuneresponse generated against a protein antigen mixed with him; being thiseffect dependent on the expression of one or several proteins coded bythe DNA, in the immunized host. The DNA is obtained from a bacterialstrain and purified according to traditional procedures (Horn et al., HGene Ther 1995, 6, 565-573).

[0021] The vaccine formulation comprises in preferred embodiments atleast one of the following plasmids: plDKE1S, plDKE2 and PAEC-ME, whoseDNA sequences coding for the protein variants expressed are identifiedwith the number of sequence of 2-4, respectively.

[0022] The plDKE1S plasmid, expressed a protein that comprise the aafrom the 176 to the 363 of the HCV E1 (Sec. Id. No. 2). On the otherhand, the plDKE2 plasmid expressed a protein encompassing the first 650aa of the viral polyprotein (C, E1 and a part of the E2) (Sec. Id.No.3). The pAEC-ME plasmid expresses a chimeric protein comprising B andT cells epitopes of different HCV antigens (Sec. Id. No. 4). In theseplasmids, the coding sequence for the viral antigens was obtained fromthe cDNA of a HCV cuban isolate (Morales et al., 1998, WO 9825960). ThepAEC-ME, plDKE1S and plDKE2 plasmids contain the coding sequence for theHCV antigens inserted into the multiple cloning site of the pAEC-K6plasmid (Herrera et al., Biochem Biophys Res Commun. 2000, 279,548-551). The plasmids included in the present invention have theregulatory elements able to direct the antigen expression in humancells. These regulatory elements include a transcriptional unitfunctional in mammals, integrated for example by the humancytomegalovirus promoter and the polyadenilation signal of the simianvirus 40. These plasmids also contain a replication origin in bacteriaand a selection marker for the resistance to kanamyicin.

[0023] The protein component of the formulation can be a soluble viralantigen able to form particles, with a purity superior to 90%. Inpreferred embodiments, are component of the vaccine formulation thecapsid antigens of the HCV and HBV, that enhanced the immune responsegenerated against the proteins expressed by the DNA mixed with them.

[0024] The present invention also contemplates the procedure for themixture of the DNA with the antigen. The mixture is prepared by additionof components, DNA and antigen, dissolved in an appropriate buffer. Inpreferred embodiments, the formulation can be prepared by thecombination of both components, dissolved in saline phosphate, in 101(ww) proportion. The mixture is incubated at least 2 h between 26° C.and 30° C., with shaking, before administration to the individuals. Thisformulation can be administered by intramuscular, subcutaneous,intraperitoneal, intramucosal, intravenous sublingual way, or others.The immunization can be performed by means of syringes, gene gun, spraysor other delivery devices. Each individual receives a dose ranging from0.001 to 10 mg of each component in a volume determined by the animalspecies and the immunization method employed. In the case of vaccineformulations having as components a DNA mixed with a protein antigen, asuperior product can be obtained compared with each one of theindividual components due to:

[0025] It is possible to generate a stronger and diverse both humoraland cellular immune response directed to a broader range of epitopes.

[0026] The toxic effect generated by the injection of the adjuvant canbe eliminated because the antigen 2 is simultaneously the adjuvant.

[0027] It is possible the employment of these formulations as core forcombined vaccines.

[0028] The process of vaccine formulation doesn't require of adsorptionof the antigen.

[0029] In the case of the formulations containing a DNA that expressed aprotein variant that include regions of the HCV E1 protein, and the HCVcapsid protein, a superior product can be obtained compared with eachone of the individual components due to:

[0030] It is possible to generate a stronger and diverse both humoraland cellular immune response directed to a broader range of epitopes.

[0031] The toxic effect generated by the injection of the adjuvant canbe eliminated because the antigen 2 is simultaneously the adjuvant.

[0032] It is possible the employment of the DNA plus the capsid as corefor combined or multivalent vaccines.

[0033] On the other hand, the immunization with a DNA that expresses aprotein variant that includes regions of the HCV E1 protein increasedthe immunogenicity of HBV protein antigens, present in the formulation.Particularly, the mixture with the HBsAg or the HBcAg, allows superiorresults to those obtained with this antigens due to:

[0034] a) The levels of IgG induced against the HBsAg are superior tothose obtained with the inoculation of the HBsAg with aluminumhydroxide.

[0035] b) Constitutes a potential combined vaccine HBV-HCV.

[0036] c) The formulation process doesn't require of adsorption of theantigen.

DESCRIPTION OF THE FIGURES

[0037]FIG. 1: Schematic representation of the plasmids pAEC-ME, plDKE1Sand plDKE2.

[0038]FIG. 2: E1ectron microscopy of the particles of the hepatitis Cvirus capsid (A), of the hepatitis B virus surface antigen (B) and ofthe hepatitis B virus capsid (C).

[0039]FIG. 3: Immunization schedule with the plDKE2 plasmid and the Coreprotein. The animals were immunized intramuscularly with 50 μg of DNAand 5 μg of protein.

[0040]FIG. 4: Immunization schedule with different plasmids and theprotein HBcAg. The animals were immunized intramuscularly with 50 μg ofDNA and 5 μg of protein.

[0041]FIG. 5: Immunization schedule with different plasmids and theprotein HBsAg. The animals were immunized intramuscularly with 50 μg ofDNA and 5 μg of protein.

EXAMPLES Example 1 Immunogenicity of Formulations Having as Components aDNA that Expresses a Polyprotein Capsid-E1-E2 of the HCV, and theProtein Antigen of the HCV Capsid.

[0042] With the objective of demonstrating the enhancement of the immuneresponse generated against the HCV structural antigens after theadministration of the mixture of the plasmid plDKE2 (FIG. 1), withrecombinant HCV capsid particles (FIG. 2A), 10 BALBc females mice, 8weeks old, per group were inoculated intramuscularly. The scheduleincluded 2 inoculations in the days 0 and 21, except one of the groupsin which the influence of a single dose in day 0 was studied. Bloodsamples were taken 14 weeks after the first immunization. Immunogenswere administered in phosphate buffer saline (PBS). The group 1 wasinoculated with 50 μg of the plDKE2 plasmid (FIG. 1, the plasmidcontains the sequence coding for the first 650 aa of the viralpolyprotein, Sec. Id. No.3). The group 2 was inoculated with 5 μg of theCore protein (comprising the first 173 aa of the HCV capsid protein).The group 3 received a first dose with 5 μg of the Core protein and asecond one with 50 μg of the plDKE2 (CoreplDKE2). The group 4 wasinoculated under similar conditions to the group 3 but in inverse order(plDKE2Core). The group 5 was inoculated with the mixture of 50 μg ofthe plDKE2 and 5 μg of the Core protein in the days 0 and 21(CoreplDKE2). The group 6 was inoculated in the same way that the group5 but only in the day 0 (CoreplDKE2 (1)). Additionally, a seventh group,negative control, was immunized with 50 μg of the plasmid pAEC-K6 (itdoesn't contain sequences coding for the HCV antigens).

[0043] The antibody response was determined by ELISA to detect the Abresponse against the HCV structural proteins. The Student T test wasemployed to analyze the results, statistical differences were consideredfor p <0.05.

[0044] The FIG. 3 shows that it is possible to increase the immuneresponse against the HCV structural antigens by the administration oftwo doses of the mixture of the plDKE2 with the Core protein withrespect to the individual components. This formulation (in two doses)induced Ab titers against the HCV E1 and E2 envelope proteinsstatistically higher to those obtained in the remaining groups (FIG.3A). These Ab titers were also statistically higher to the levels of Absagainst the HCV capsid protein, generated by the plDKE2-Core mixtureadministered in a single dose (FIG. 3A). The inoculation of the mixturein a single dose always induced the lower levels of Abs among theimmunized groups.

[0045] The evaluation of the lymphoproliferative response against theHCV structural antigens (FIG. 3B) indicated a significantly superiorresponse against the capsid in the group of animals immunized with theplDKE2-core in 2 doses, with respect to the remaining groups. Theresults are shown as the stimulation index of spleen cells obtained fromimmunized animals. The stimulation index was determined by the (H³)Thymidine uptake. It is possible to conclude that the immunization withthe mixture of plDKE2 and the Core protein generates a synergicstimulation of the immune response induced against the HCV structuralantigens.

Example 2 Immunogenicity of Formulations Having as Components a DNA thatExpresses a Polyprotein Capsid-E1-E2 of the HCV, and the Protein Antigenof the HBV Capsid.

[0046] With the objective of investigating the behavior of the immuneresponse generated by the mixture of the plDKE2 plasmid with proteinantigens of other pathogens, 10 females BALBc/mice, 8 weeks old, pergroup were inoculated intramuscularly with the mixture of the abovereferred plasmid with recombinant particles of the HBV capsid (HBcAg,FIG. 2C). The schedule included 2 inoculations in the days 0 and 21.Blood samples were taken at 9 and 19 weeks after the first immunization.Immunogens were prepared in phosphate buffer saline (PBS). The plasmidswere administered in dose of 50 μg , and the HBV capsid protein in doseof 5 μg . The group 1 was inoculated with the plasmid pAEC-K6 (negativecontrol). The group 2 was administered with the HBcAg protein. The group3 was vaccinated with plDKE2. The groups 4 and 5 were vaccinated withthe mixture of the HBcAg with the plasmids plDKE2 and pAEC-K6,respectively. The Student T test was employed to analyze the resultsstatistically, a significant difference was considered for p<0.05.

[0047] The FIG. 4 shows the antibody response induced in mice 19 weeksafter primary immunization. FIG. 4A shows that the mixture of the plDKE2plasmid with the HBcAg induced Ab titers against the HBcAg,statistically higher to the observed in the rest of the vaccinatedanimals. No statistical differences were detected between the groupsimmunized with HBcAg alone or mixed with the pAEC-K6. Therefore it ispossible to conclude that the plasmid plDKE2 enhance the immune responseagainst the HBcAg.

[0048] On the other hand, the FIG. 4B shows that the mixture of theplDKE2 plasmid with the HBcAg induces antibody titers against the HCVstructural antigens higher to those generated in the animals immunizedwith the plDKE2 alone. Therefore, the HBcAg is also capable of enhancethe immune response induced against the HCV structural antigens inducedafter the administration of the plDKE2.

Example 3 Immunogenicity of Formulations Having as Components PlasmidsExpressing Variants of the HCV and HBV, and the Protein Variant of theHBV Surface Antigen.

[0049] With the objective of demonstrating the enhancement of the immuneresponse generated against other protein antigens observed after theco-administration with the plDKE2 plasmid, and to study other plasmidswith similar adjuvant properties, 10 female BALBc mice, 8 weeks old, pergroup were inoculated intramuscularly with the mixture of the plasmidwith recombinant particles of the HBsAg (FIG. 2B). The schedule included3 inoculations in days 0, 21 and 50. Blood samples were taken at week16, after the primary immunization. All the immunogens were prepared inphosphate buffer saline (PBS), except a group formulated with Aluminumhydroxide. The group 1 was inoculated with the mixture of 50 μg of theplasmid plDKCo, containing the sequence coding for the first 176 aa ofthe HCV capsid protein (Dueñas-Carrera et al., Vaccine2000;19(7):992-997), and 5 μg of the HBsAg (plDKCo-HBsAg). The groups 2to 7 were inoculated with mixtures of DNA and HBsAg in same quantitiesbut using the following plasmids: group 2 (plDKE1S-HBsAg), the plasmidplDKE1S (FIG. 1, containing the sequence coding for the aa 176-363 ofthe HCV polyprotein, Sec. Id. No.2); group 3 (pAEC-ME-HBsAg), theplasmid pAEC-ME (FIG. 1, containing the sequence coding for a proteinthat includes different epitopes of the HCV antigens, Sec. Id. No.4);group 4 (plDKE2-HBsAg), the plasmid plDKE2 (FIG. 1) containing thesequence coding for the aa 1-650 of the HCV polyprotein, Sec. Id. No.3;group 5 (plDKE1Sm-HBsAg), the plasmid plDKE1Sm is identical to theplDKE1S except that it includes 2 nucleotide insertions in the regioncoding for the HCV E1 that changes the open reading frame and impedesthe expression of this protein (Sec. Id. No.5); group 6(pAEC-d2-HBsAg-HBsAg), the plasmid pAEC-d2-HBsAg contains the sequencecoding for the HBV HBsAg (Musacchio et al., Biochem Bioph Res Commun2001, 282, 442446); group 7 (pAEC-K6-HBsAg), the plasmid pAEC-K6(negative control, doesn't contain coding sequence under the control ofthe transcriptional unit). Finally, the groups 8 and 9 were inoculatedwith 5 μg of HBsAg formulated in Aluminum hydroxide or alone,respectively. The Student T test was employed to analyze the resultsstatistically, a significant difference was considered for p <0.05.

[0050] The FIG. 5 shows the Abs titers generated against the HBsAg, 16weeks after primary immunization. The levels of Abs induced by the HBsAgalone in PBS were statistically inferior to the rest of the variantsevaluated except for the mixture formed by the HBsAg and the pAEC-K6. Onthe other hand, the mixtures of HBsAg with the plasmids plDKCo, plDKE1S,pAEC-ME and plDKE2 induced Ab titres against the HBsAg statisticallyhigher to those induced by the immunization with the HBsAg formulated inAluminum hydroxide or mixed with the pAEC-K6. The immunization with theHBsAg formulated with aluminum hydroxide or mixed with pAEC-K6, plDKE1Smand pAEC-d2-HBsAg induced similar levels of Ab titers against the HBsAg.It is possible to conclude that the expression in the host cells ofprotein variants that include the amino acid regions of the HCV E1antigen, from the plasmids administered, enhance the immune responsegenerated against the protein antigen mixed with the DNA construct.

1 5 1 531 DNA Artificial sequence gene (1)..(528) Includes the sequencecoding for aa 1 to 176 of the HCV core protein 1 atgagcacga atcctaaacctcaaagaaaa accaaacgta acaccaaccg ccgcccacag 60 gacgtcaagt tcccgggcggtggtcagatc gttggtggag tttacctgtt gccgcgcagg 120 ggccccaggt tgggtgtgcgcgcaactagg aagacttccg agcggtcgca acctcgtgga 180 aggcgacaac ctatccccaaggctcgccgg cccgagggca ggtcctgggc ccagcccggg 240 tacccttggc ccctctatggtaacgagggc atgggatggg caggatggct cctgtcaccc 300 cgtggctctc ggcctagttggggccccact gacccccggc gtaggtcgcg taatttgggt 360 aaggtcatcg ataccctcacatgcggcttc gccgacctca tggggtacat tccgctcgtc 420 ggcgcccccc tagggggcgctgccagggcc ctggcgcatg gcgtccgggt tctggaggac 480 ggcgtgaatt atgcaacagggaatctgccc ggttgctctt tctctctcta a 531 2 567 DNA Artificial sequencegene (1)..(564) Includes the nucleotide sequence coding for the aa176-363 on HCV polyprotein, mainly corresponding to the E1 protein. 2atgttccttt tggctttgct gtcctgtttg accatcccag tttccgccta tgaagtgcgc 60aacgcgtccg gggtgtacca tgtcacgaac gactgctcca actcaagcat tgtgtatgag 120gcagacgaca tgatcatgca cacccccgga tgcgtgccct gcgttcggga ggacaacacc 180tcccgctgct gggtagcgct cacccccaca ctcgcggcca ggaatgccag cgtccccacc 240acgacaatac gacgccacgt cgatttgctc gttggggcgg ctgctctctg ctccgctatg 300tacgtggggg atctctgcgg atctgttttc ctcgtttccc agctgttcac cttctcgcct 360cgccggcatg agacagcaca ggactgcaac tgctcaatct atcccggcca cgtatcaggt 420caccgcatgg cctgggatat gatgatgaac tggtcacctt caacagccct agtggtatcg 480cagttactcc ggatcccaca agccgtcgtg gacatggtag cgggggccca ctggggagtc 540ctagcgggcc ttgcctacta ctcctaa 567 3 1953 DNA Artificial sequence gene(1)..(1950) Includes the nucleotide sequence coding for aa 1-650 on HCVpolyprotein, encompassing the capsid, E1 and a portion of the E2protein. 3 atgagcacga atcctaaacc tcaaagaaaa accaaacgta acaccaaccgccgcccacag 60 gacgtcaagt tcccgggcgg tggtcagatc gttggtggag tttacctgttgccgcgcagg 120 ggccccaggt tgggtgtgcg cgcaactagg aagacttccg agcggtcgcaacctcgtgga 180 aggcgacaac ctatccccaa ggctcgccgg cccgagggca ggtcctgggcccagcccggg 240 tacccttggc ccctctatgg taacgagggc atgggatggg caggatggctcctgtcaccc 300 cgtggctctc ggcctagttg gggccccact gacccccggc gtaggtcgcgtaatttgggt 360 aaggtcatcg ataccctcac atgcggcttc gccgacctca tggggtacattccgctcgtc 420 ggcgcccccc tagggggcgc tgccagggcc ctggcgcatg gcgtccgggttctggaggac 480 ggcgtgaatt atgcaacagg gaatctgccc ggttgctctt tctctctcttccttttggct 540 ttgctgtcct gtttgaccat cccagtttcc gcctatgaag tgcgcaacgcgtccggggtg 600 taccatgtca cgaacgactg ctccaactca agcattgtgt atgaggcagacgacatgatc 660 atgcacaccc ccggatgcgt gccctgcgtt cgggaggaca acacctcccgctgctgggta 720 gcgctcaccc ccacactcgc ggccaggaat gccagcgtcc ccaccacgacaatacgacgc 780 cacgtcgatt tgctcgttgg ggcggctgct ctctgctccg ctatgtacgtgggggatctc 840 tgcggatctg ttttcctcgt ttcccagctg ttcaccttct cgcctcgccggcatgagaca 900 gcacaggact gcaactgctc aatctatccc ggccacgtat caggtcaccgcatggcctgg 960 gatatgatga tgaactggtc accttcaaca gccctagtgg tatcgcagttactccggatc 1020 ccacaagccg tcgtggacat ggtagcgggg gcccactggg gagtcctagcgggccttgcc 1080 tactactcca tggtggggaa ctgggccaag gttttgattg tgatgctactctttgccggc 1140 gttgacggga cgggaaccta cgtgacaggg gggacggcag cccgcggcgtcagccagttt 1200 acgggcctct ttacatctgg gccgagtcag aaaatccagc ttgtaaataccaacggcagc 1260 tggcatatta accggactgc cctgaactgc aacgactccc tccagaccgggttccttgct 1320 gcgttgtttt acgtgcacag gttcaactcg tccggatgct cagatcgcatggccagctgc 1380 cgccccattg atacgttcga ccaggggtgg ggccccatta cttacgctgagccgcgcagc 1440 ttggaccaga ggccctattg ctggcactac gcacctcaac cgtgtggtatcgtacccgcg 1500 gcggaggtgt gtggtccagt gtattgtttc actccaagcc ccgttgtcgtggggaccacc 1560 gatcgttccg gcgtccctac gtataactgg ggggagaatg agacggacgtgctgctcctt 1620 aacaacacgc ggccgccgct gggcaactgg tttggctgta catggatgaatagcactggg 1680 ttcaccaaga cgtgcggggg ccctccgtat aacatcggag gggtcggtaacaacaccttg 1740 acctgcccta cggattgctt ccgcaagcac cccgaggcca cttacaccaaatgtggtttg 1800 gggccttggt tgacacctag gtgcttggtc gactacccat acaggctttggcattacccc 1860 tgcactgtca actttaccat cttcaaggtt cggatgtatg tggggggcgtggagcacagg 1920 cttaccgctg catgcaactg gactcgagga taa 1953 4 1194 DNAArtificial sequence gene (1)..(1191) Includes the nucleotide sequencecoding for different epitopes of HCV proteins. 4 atgacgggaa cctacgtgacaggggggacg gcagcccgcg gcgtcagcca gtttacgggc 60 ctctttacat ctgggccgagtcagaaaatc cagcttgtaa ataccaacgg cagctggcat 120 attaaccgga ctgccctgaactgcaacgac tccctccaga ccgggttcct tgctgcgttg 180 ttttacgtgc acaggttcaactcgtccgga tgctcagatc gcatggccag ctgccgcccc 240 attgatacgt tcgaccaggggtggggcccc attacttacg ctgagccgcg cagcttggac 300 cagaggccct attgctggcactacgcacct caaccgtgtg gtatcgtacc cgcggcggag 360 gtgtgtggtc cagtgtattgtttcactcca agccccgttg tcgtggggac caccgatcgt 420 tccggcgtcc ctacgtataactggggggag aatgagacgg acgtgctgct ccttaacaac 480 acgcggccgc cgctgggcaactggtttggc tgtacatgga tgaatagcac tgggttcacc 540 aagacgtgcg ggggccctccgtataacatc ggaggggtcg gtaacaacac cttgacctgc 600 cctacggatt gcttccgcaagcacggatcc acccacgtga ccggcggcag ccaggcccgc 660 accacccaca gcttcacctccctgctgcgc cagggcgcca agcagaacgt gcagctgatc 720 gccgacctga tgggctacatcccactggtg ggcgccccac tgggcaagaa gggccacgtg 780 agcggccacc gcatggcctgggacatgatg atgaactggg ccagcaagaa ggccgccagc 840 cgcgccgccg gcttgcaggacagcaccatg ctggtgagcc acacccgcgt gaccggcggc 900 gtggccggcc acgtgaccagcggcctggtg tccctgttca gccctggcgc cagccagaag 960 atccagctgg tgggctccagcttcagcctg ttcctgttgg ccctcctgag cagcttgacc 1020 atcaagaaga tgagctactcctggaccggc gccctggtga ccccaagcgc cgccgagaag 1080 aagctgttgt tcaacatcctgggcggctgg gtgaagaaga gcatggtggg caactgggcc 1140 aaggtgaaga agtacaccggcgacttcgac agcgtgatcg actccaggcc ttaa 1194 5 569 DNA Artificial sequenceDescription of the artificial sequence pIDKE1Sm 5 attgttcctt ttggctttgctgtcctgttt gaccatccca gtttccgcct atgaagtgcg 60 caacgcgtcc ggggtgtaccatgtcacgaa cgactgactc caactcaagc attgtgtatg 120 aggcagacga catgatcatgcacacccccg gatgcgtgcc ctgcgttcgg gaggacaaca 180 cctcccgctg ctgggtagcgctcaccccca cactcgcggc caggaatgcc agcgtcccca 240 ccacgacaat acgacgccacgtcgatttgc tcgttggggc ggctgctctc tgctccgcta 300 tgtacgtggg ggatctctgcggatctgttt tcctcgtttc ccagctgttc accttctcgc 360 ctcgccggca tgagacagcacaggactgca actgctcaat ctatcccggc cacgtatcag 420 gtcaccgcat ggcctgggatatgatgatga actggtcacc ttcaacagcc ctagtggtat 480 cgcagttact ccggatcccacaagccgtcg tggacatggt agcgggggcc cactggggag 540 tcctagcggg ccttgcctactactcctaa 569

We claim:
 1. A vaccine formulation having as components, a DNA thatexpresses a protein and a protein antigen, wherein at least one of thecomponents of this formulation acts as adjuvant of the other one.
 2. Avaccine formulation according to claim 1, having as components, a DNAthat expresses a protein variant that includes regions of the hepatitisC virus E1 antigen, and a viral protein.
 3. A vaccine formulationaccording to claim 1, having as components, a DNA identified by the SEQID No. 3, and the hepatitis C virus capsid protein.
 4. A vaccineformulation according to claim 1, having as components a DNA identifiedby the SEQ ID No. 3, and the hepatitis B virus capsid protein.
 5. Avaccine formulation according to having as components a DNA identifiedby the SEQ ID No. 2 to 4, and the hepatitis B virus surface antigen. 6.A vaccine formulation according to claim 1, employed as a therapeuticand/or preventive agent against the hepatitis C virus.
 7. A vaccineformulation according to claim 4, to be employed as a therapeutic and/orpreventive agent against the hepatitis B virus.
 8. A vaccine formulationaccording to claim 4, to be employed as a therapeutic and/or preventiveagent against both the hepatitis C virus and the hepatitis B virus.
 9. Avaccine formulation according to claim 2, having as components, a DNAidentified by the SEQ ID No. 3, and the hepatitis C virus capsidprotein.
 10. A vaccine formulation according to claim 2 having ascomponents a DNA identified by the SEQ ID No. 3, and the hepatitis Bvirus capsid protein.
 11. A vaccine formulation according to claim 2,having as components a DNA identified by the SEQ ID No. 2 to 4, and thehepatitis B virus surface antigen.
 12. A vaccine formulation accordingto claim 2, to be employed as a therapeutic and/or preventive agentagainst the hepatitis C virus.
 13. A vaccine formulation according toclaim 11, to be employed as a therapeutic and/or preventive agentagainst the hepatitis B virus.
 14. A vaccine formulation according toclaim 11, to be employed as a therapeutic and/or preventive agentagainst both the hepatitis C virus and the hepatitis B virus.